One-Pot Access to Functionalised Malamides via Organocatalytic Enantioselective Formation of Spirocyclic β-Lactone-Oxindoles and Double Ring-Opening

Malamides (diamide derivatives of malic acid) are prevalent in nature and of significant biological interest, yet only limited synthetic methods to access functionalised enantiopure derivatives have been established to date. Herein, an effective synthetic method to generate this molecular class is developed through in situ formation of spirocyclic β-lactone-oxindoles (employing a known enantioselective isothiourea-catalysed formal [2+2] cycloaddition of C(1)-ammonium enolates and isatin derivatives) followed by a subsequent dual ring-opening protocol (of the β-lactone and oxindole) with amine nucleophiles. The application of this protocol is demonstrated across twelve examples to give densely functionalised malamide derivatives with high enantio- and diastereo-selectivity (up to >95:5 dr and >99:1 er).


Introduction
Malic acid (2-hydroxybutanedioic acid) is a readily available platform chemical that is widely used in the food and cosmetic industries [1][2][3][4].It is readily available in an enantiomerically pure form and has been utilised as a versatile C(4)-synthetic building block for a range of natural product syntheses [5].Amide derivatives of malic acid (generally known as malamides) are prevalent in nature [6][7][8][9][10] and are of significant biological interest, as they are isosteric to asparagine and aspartic acid and have been used against HCV infections [11] and as a constituent of agents that provide DNA binding to p53 [12].They can also be used as metalating reagents [13,14] and serve as metalloprotease inhibitors [15][16][17][18][19][20] or as peptidomimetics (Figure 1A) [13,17,[21][22][23]].As a representative example, DuPont Merck developed a chiral auxiliary-based route to a range of matrix metalloproteinase (MMP) inhibitors based upon the malamide motif, with the absolute configuration at C(2) essential for bioactivity [17].Despite this interest, catalytic enantioselective protocols to access bespoke highly functionalised malamide derivatives with high stereocontrol are generally lacking but could be of widespread interest to the synthetic community.

Results
Building upon the literature precedent, initial studies probed the hypothesis that changing from an N-alkyl-protected isatin to incorporating a conjugating electron-withdrawing N-substituent would facilitate double ring-opening after enantioselective [2+2] cycloaddition.Initially, an N-phenyl substituent was considered, with N-phenyl isatin treated under the previously optimised reaction conditions for the [2+2] addition of C(1)-ammonium enolates to N-alkyl isatins [11a], followed by the addition of benzylamine (Table 1, entry 1).Unfortunately, no malamide is observed, and only the ring-opening of the isatin starting material is observed (≈30% yield by 1 H NMR spectroscopic analysis).Unfortunately, the installation of an N-C(O)NHPh substituent led to the resulting isatin starting material being insoluble under the reaction conditions, returning only the starting material (entry 2).While the use of N-Ts protected isatin led to a complex product mixture (entry 3), N-Bocsubstituted isatin gave the desired malamide product 1 resulting from double ring-opening in a good 67% yield and with excellent stereocontrol (entry 4, >95:5 dr, 99:1 er) when 3.0 equivalents of benzylamine were applied.Interestingly, when the 1.0 equivalent of benzylamine was used to investigate the potential of sequential ring-opening, only the double ring-opened product 1 was obtained in 32% yield with high stereoselectivity maintained (entry 5, >95:5 dr, 98:2 er).recent work, the isothiourea HyperBTM promoted the in situ formation of β-lactone spirocyclic oxindoles via a [2+2] cycloaddition of C(1)-ammonium enolates to N-alkyl isatin derivatives, with selective β-lactone ring-opening facilitating product isolation [30].In this work, the use of N-acyl isatins facilitates an enantioselective route to spirocyclic isatinderived β-lactones, with double ring-opening (of the β-lactone and oxindole motifs) revealing the linear and densely functionalised malamide structure, including two contiguous stereogenic centres (Figure 1C).To the best of our knowledge, while reports of selectively opening either β-lactones or (spiro)oxindoles [111][112][113][114] have been disclosed previously, this double ring-opening strategy has hitherto been unknown.

Results
Building upon the literature precedent, initial studies probed the hypothesis that changing from an N-alkyl-protected isatin to incorporating a conjugating electron-withdrawing N-substituent would facilitate double ring-opening after enantioselective [2+2] cycloaddition.Initially, an N-phenyl substituent was considered, with N-phenyl isatin treated under the previously optimised reaction conditions for the [2+2] addition of C(1)ammonium enolates to N-alkyl isatins [11a], followed by the addition of benzylamine (Table 1, entry 1).Unfortunately, no malamide is observed, and only the ring-opening of the isatin starting material is observed (≈30% yield by 1 H NMR spectroscopic analysis).Unfortunately, the installation of an N-C(O)NHPh substituent led to the resulting isatin starting material being insoluble under the reaction conditions, returning only the starting material (entry 2).While the use of N-Ts protected isatin led to a complex product mixture (entry 3), N-Boc-substituted isatin gave the desired malamide product 1 resulting from double ring-opening in a good 67% yield and with excellent stereocontrol (entry 4, >95:5 dr, 99:1 er) when 3.0 equivalents of benzylamine were applied.Interestingly, when the 1.0 equivalent of benzylamine was used to investigate the potential of sequential ring-opening, only the double ring-opened product 1 was obtained in 32% yield with high stereoselectivity maintained (entry 5, >95:5 dr, 98:2 er).Subsequent work focused on probing the generality of this process to make a range of substituted malamide derivatives (Figure 2).The capability of alternative primary and secondary amines towards the double ring-opening process was investigated, with good product yields and excellent levels of enantio-and diastereoselectivity (>95:5 dr, >98:2 er) observed with benzylamine, (2-pyridylmethyl)amine and morpholine (to give 1, 2 and 3, a A total of 30% product derived from direct ring-opening of the isatin starting material was observed by analysis of the 1 H NMR spectra of the crude reaction mixture.b Yield refers to isolated product yield after chromatographic purification.c Enantiomeric ratios (er) were determined by HPLC analysis on a chiral stationary phase.Diastereomeric ratios (dr) were obtained by 1 H NMR analysis of the isolated product mixture.

Discussion
The proposed mechanism for the formation of malamide derivatives involves the previously reported enantioselective [2+2] cycloaddition strategy that is initiated through attack of the isothiourea Lewis base organocatalyst HyperBTM to phenylacetic anhydride, giving acyl ammonium species 13 (Figure 3).Deprotonation by the carboxylate counterion generates the C(1)-ammonium enolate intermediate 14 [36], which, based on previous computational studies with CF 3 -ketones, undergoes a concerted asynchronous [2+2] cycloaddition with N-Boc isatin via pre-transition state intermediate 14.Stereoselectivity is induced by the stereodirecting Ph group derived from the catalyst blocking the approach of the electrophile from the Re-face, with the enolate geometry rigidified by a n O →σ* S-C chalcogen bond [116][117][118][119][120][121][122][123].The β-lactol intermediate 15 then collapses to regenerate the isothiourea catalyst HyperBTM and the corresponding spirocyclic β-lactone-oxindole 16.Upon addition of the nucleophilic amine (illustrated using benzylamine), it is assumed that ring-opening of the β-lactone is favoured to initially give 17.From 17, two potential pathways to product 1 can be envisaged.Simplistically, ring-opening of 17 may lead directly to 1.However, a potential explanation for the observation that only double ring-opened malamide products are obtained when using one equivalent of benzylamine, even though an excess of β-lactone is present, could involve in situ intramolecular ring-opening of the oxindole motif from 17, leading to a reactive pyrrolidindione intermediate 18, that in turn is ring-opened to generate the observed malamide product 1.We are currently unable to discriminate between these potential reaction pathways, but both lead to the observed product.

Materials and Methods
All reagents and solvents were obtained from commercial suppliers and were used without further purification unless otherwise stated.Anhydrides and N-Boc isatin were prepared in accordance with the literature [30,124].Reactions involving moisture-sensi-

Materials and Methods
All reagents and solvents were obtained from commercial suppliers and were used without further purification unless otherwise stated.Anhydrides and N-Boc isatin were prepared in accordance with the literature [30,124].Reactions involving moisture-sensitive reagents were carried out in flame-dried glassware under an inert atmosphere (N 2 or Ar) using standard vacuum line techniques.Anhydrous and ethanol-free CH 2 Cl 2 was obtained after passing through an alumina column (MBraun SPS-800).Analytical thin layer chromatography (TLC) was performed on pre-coated aluminium plates (Kieselgel 60 F254 silica), and visualisation was achieved using ultraviolet light (254 nm) and/or staining with aqueous KMnO 4 solution by heating.Manual column chromatography was performed in glass columns fitted with porosity 3 sintered discs over Kieselgel 60 silica using the solvent system stated.

Conclusions
In conclusion, this manuscript demonstrates an effective one-pot process for the synthesis of stereodefined functionalised malamides through employing an enantioselective organocatalytic [2+2]-cycloaddition of C(1)-ammonium enolates with isatin derivatives and consecutive double ring-opening of the corresponding β-lactone-spirooxindole.The malamide products can be isolated in moderate to good yields (28% to 84% yield) and excellent enantio-and diastereo-selectivity (up to >95:5 dr, >99:1 er).This route exemplifies a simple and efficient one-pot synthetic protocol to access enantiomerically pure malamide materials using isothioureas as Lewis base catalysts.

i 1 Figure 3 .
Figure 3. Illustrative proposed reaction mechanism for the generation of stereodefined malamide derivatives.